All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
Pre-designed self-assembling scaffolds are highly advantageous in areas such as tissue regeneration/engineering, 3D cell culture, in vitro toxicity testing, understanding cell/extracellular matrix interactions, controlled stem cell differentiation, studies of mechanical loading effects on cells, and the study of metastasis models. In particular, developments in the use of self-assembling peptides provide potential for the use of such novel bionanomaterials in tissue engineering. The various properties of the amino acids in peptides, their biological compatibility, and the inherent properties of their bonded structure make peptides a very powerful building block for the fabrication of self-assembling scaffolds.
Advances have been made in creating synthetic mimics of the Extracellular Matrix for in vivo and in vitro applications. Some researchers have described the use of peptides with alternating charged, hydrophobic and hydrophilic amino acids to culture nerve cells, endothelial cells and chondrocytes. Other researchers have demonstrated the use of synthetic amphiphile peptide-containing molecules that can self-assemble into fibrous scaffolds that support cell growth and stem cell differentiation. These successes illustrate that man-made hydrogels could be useful for forming scaffold materials for 3D cell culture and tissue engineering applications.
Xu et. al. (J. Am. Chem. Soc. 2003, 125, 13680) described that Fmoc (fluorenylmethoxycarbonyl) protected di-peptides could form fibrous scaffolds at low pH values by taking advantage of π-stacking of the highly conjugated Fmoc group. Examples of Fmoc-dipeptides disclosed by Xu et al as being capable of forming such gels are Fmoc-D-Ala-D-Ala (3), Fmoc-L-Ala-L-Ala (3), Fmoc-Gly-Gly (3), Fmoc-Gly-D-Ala (5) and Fmoc-Gly-L-SER (5), the numbers in parentheses being the pH value for gelation. Fmoc is widely used as a protecting group in peptide chemistry and when coupled to amino acids, is known to have anti-inflammatory properties, as demonstrated in animal studies. The Fmoc group acts as a “stacking ligand”, thought to offer order and directionality to the self-assembly process. However, Xu et al carried out all of their investigations at substantially acidic pH's (i.e. pH 3-5), and did not investigate whether the compounds could be used in biologically acceptable (ie. physiologically agreeable) conditions.
Although considerable efforts have been made towards understanding the behaviour of hydrogel scaffolds, the present knowledge on the subject is very limited as much of these studies have been based on trial and error. Furthermore, little has been reported on the rules that govern self-assembly or the functioning of the peptide scaffolds under different conditions. For use of such scaffolds in biological or medical conditions, it is important to understand the scaffold behaviour, especially under environmental conditions similar to those experienced in vivo. Furthermore, ultimately, researchers would like to design scaffolds rationally for use in vivo.
Furthermore, up until now, it has not been possible to make scaffolds from small molecule building blocks that are: (i) stable under tissue culture conditions (i.e. high ionic strength, and pH 7); (ii) of similar dimensions to fibrous components of the extracellular matrix; (iii) capable of supporting cell culture in 3D; (iv) optically transparent; and (v) capable of liquid to gel transitions on demand by biocompatible means.
Therefore, it is an aim of the present invention to obviate or mitigate one or more of the problems of the prior art, whether identified herein or elsewhere, and to provide improved hydrogels, which may be used in vitro or in vivo to support cell cultures, and to provide methods of treatment, which use such hydrogels.